Ovarian aging permanently alters the female hormone profile, causing sequellae such as infertility, osteoporosis, and cardiovascular disease. Before menopause, age-related changes in hormone production occur during both phases of the menstrual cycle. When compared to younger counterparts, older women have lower levels of luteal phase hormones, which are produced by the corpus luteum (CL). Unfortunately, the mechanism of age-related CL dysfunction is unknown. Ovarian aging is a fibrotic process involving dramatic stromal remodeling, which increases the microenvironment rigidity. While it is known that ECM- derived signals regulate CL steroidogenesis, the causative link between microenvironment rigidity and hormone production has not previously been shown. Thus, in the studies proposed herein, we will test the hypothesis that changes in the ovarian physical environment cause progressive luteal phase hormone decline in an aging mouse model. In preliminary studies, we have cultured murine CLs in a novel 3D alginate system, which can be tuned to different mechanical rigidities. In Aim 1, proposed experiments will manipulate alginate rigidity and define the effects on CL structure and steroidogenesis. Specifically, experiments will phenocopy older CLs by culturing glands from younger animals in more rigid alginate conditions. Conversely, a rescue experiment will be performed with CLs from older animals cultured in less rigid alginate environments. In addition, the relationship of ECM adhesivity and mechanical force will be explored by competitively inhibiting integrin binding with free RGD peptides. In the second Aim of this proposal, experiments will test a possible mechanism of luteal cell mechanotransduction. In many cell types, Rho/ROCK signaling plays a chief role in sensing the physical milieu and is a known effector of ECM-integrin engagement. Moreover, Rho/ROCK signaling could be the critical link between ECM rigidity, integrin binding, and luteal cell structure-function. Experiments will assess Rho/ROCK signaling in younger and older animal cohorts. Additionally, we will test Rho/ROCK signaling in CLs cultured in various rigidities using our 3D hydrogel system and several pathway inhibitors. By harnessing the ability to bioengineer the ovarian microenvironment, we can identify regulatory mechanisms at the intersection of age, tissue rigidity, and hormone production. In so doing, we will test a novel theory of female reproductive aging and identify mechanisms that may be regulated in vivo to improve luteal function in the aging, premenopausal women.